HI. My name is Yiyan Zhang, or you can call me Joycelyn.I am a senior student from China. I am in a normal school which has really limited resources of laboratory around Beijing, so I am so fascinated about all these great resources from BLI. I especially interested in Molecular Biology, and I am trying out to get the access to study in America.
While the overwhelming way to edit genome, CRISPR, allows human beings to finish permanent modification of genes within organisms, this new ability is also attracting people to explore the ways. People have used four families of engineered nucleases. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
In 2nd May, 2016, an article named DNA-guided genome editing using NgAgo (the Natronobacterium gregoryi Argonaute) came up with a new way to edit genome. Since it was first published in Nature Biotech in May 2016, NgAgo has received much attention and the plasmid has been requested by labs around the globe nearly 400 times.
Argonautes a nuclease, which exists in the bacteria, plants, fungi, mammalian cells. Recently found TtAgo acquired from bacterial Argonaute capable of binding to single-stranded DNA and the latter as a guide to degrade DNA plasmids. This phenomenon prompted Argonaute certain species can be used as internal DNA endonuclease.
Like the CRISPR/Cas system, NgAgo was reported to be suitable for genome editing, but this has not been replicated.
Today, I gonna talk around this sentence. The third sentence appears on the Wikipedia page of NgAgo.
As aimed to the same target with CRISPR, NgAgo has the similarity with CRISPR in many ways, but it also has its own advantages.
Here is a side-by-side comparison of the two systems, based on data presented in the NBT paper as well as the characteristics of each component in the gene editing systems.
1. Comparable gene editing efficiency
2. Higher fidelity (less off-target effect)
Data showed that NgAgo resulted in low guide–target mismatch and high efficiency when editing difficult templates, such as GC-rich genomic targets. Because the cells themselves produce large amounts of RNA fragments, Cas9 have with these non-specific RNAs binding possibility misguided cutting non-targeting of genomic location; and Cas9 to guide RNA and DNA sequences targeting high mismatch tolerance (5 nucleotide mismatches will not stop cutting), so there is a certain off-target Cas9 defects. Unlike Cas9 only contains 19 bases, the gDNA combined with NgAgo contains 24 bases. These 5 more bases, theoretically, can promote the accuracy 4 of 5th power, that is 1024 times.
3. Fewer sequence constraints (no need for PAM)
NgAgo did not seem to require a protospacer-adjacent motif (PAM), making it easier to use for targeted genome editing, which gives a researcher unprecedented freedom to target any sequence of DNA.
4. Guides are readily available (5’p ssDNA oligo could be purchased at low cost)
Using 5’ phosphorylated ssDNAs as guide molecules reduces the possibility of cellular oligonucleotides misleading NgAgo. A guide molecule can only be attached to NgAgo during the expression of the protein. Once the guide is loaded, NgAgo cannot swap free floating ssDNA for its gDNA.
5. Guides are of higher stability (DNA as compared to RNA in CRISPR/Cas9 system)
NgAgo target specificity is dictated by a DNA guide sequence rather than an RNA guide as with Cas9. Since the formation of RNA secondary structure easier, so rich gene sequence GC, Cas9 system inefficient
While both the CRISPR-Cas9 and NgAgo genome engineering methods have their own pros and cons.
1. Unlike an RNA guide, it cannot be produced from a plasmid in cells. 2. Additionally, in vitro assembly of NgAgo/ssDNA requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes the endonuclease activity of the enzyme. 3. In vivo, only nascent NgAgo protein (probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene in vivo.
The technology nuclease specific sites of the genome mutations, insertions, substitutions and other transformation. Gene editing process includes three steps:
First, a specially designed wizard DNA or RNA (gene-oriented element) and endonuclease (Gene cutting element) binding, here is gDNA, which first led to a specific area targeted gene;
Secondly, the nucleic acid endonuclease gene targeting specific areas for cutting, each on two DNA to form a gap, resulting in double-strand break (DSB). Here use argonaute endonuclease enzyme
Finally, the activation of innate intracellular DSB repair mechanisms of this phenomenon, the repair gap At the same time can be mutations, insertions, substitutions and other transformation.
“one-guide faithful” rule guides can only be loaded when NgAgo protein is in the process of expression
5 'end phosphorylated single-stranded DNA as a guide wizard, Argonaute nuclease combined with the Wizard DNA, for DNA complementary to the target DNA wizard cutting, resulting in the targeting DNA double-strand breaks.
Enzyme is a nucleic acid capable of Argonaute 5 'phosphorylated end of single-stranded DNA as short-chain double-stranded DNA in a targeting guide at 10-50 ° C under endonuclease cleavage of double-strand breaks caused by Argonaute nucleic acids.
After targeting genomic DNA double strand breaks caused in the presence of exogenous DNA fragments with homologous sequences of case, by the cell's own repair pathways HomologousDirectedRepair the outer source DNA fragment into double-strand breaks area, so as to achieve the targeted gene or functional areas according to the design change.
The engineered NLS-NgAgo was transfected to Hela cells and it shows that the expressed NLS-NgAgo was compartmented in the nucleus (DAPI+) and the cells maintained normal morphology.
The NgAgo replication experiments have been conducted by scientists from around the world. US, Europe, Australia, China, Japan, India
The popularity of the experiments about NgAgo also arose after the NBT article has been published. Labs and scientists are trying to repeat this new method.
Scientists have really worked hard for NgAgo, many well-established detection methods for genome editing have been utilized.
· Mammalian cell lines (HEK293, Hela, etc) · Mouse/human NS cells · Yeast · Fruit fly · C. elegans · Zebra fish · Mouse zygote
And, maybe even more…..
but this has not been replicated.
This is a global experimentation on NgAgo for genome editing; scientists from many countries have contributed budget, time and effort for it.
A group of scientists has been test NgAgo on Zebrafish, but the result from their experiments just shows that the gDNA/NgAgo system provides an alternative strategy for gene knockdown in zebrafish, but not edit the genome.
Trying to find out which part is wrong, some also analyzed the NBT paper. They got the logic in this paper in this chart.
E. coli expressed and purified GST-NgAgo needs 2 guides to linearize plasmid Mammalian expressed and purified FLAG-NgAgo-HA only needs 1 guide to linearize plasmid in vitro, E. coli GST-NgAgo needs 2 guides in vitro, Mammalian FLAG-NgAgo-HA needs only 1 guide
So in their opinion there is a jump from 2 guids to 1 guide.
Han also added A general protocol of NgAgo/gDNA-mediated genome editing trying to help for the experiments.
In his papers, he mentioned these 3 parts.
Cell culture
Transfection
Genomic DNA extraction
In conclusion,
NgAgo will have to go through a couple years of validation to inspect its safety profile. It will be interesting to see how this new system matures over time.
Research Project: NgAgo/gDNA
Powerpoint
https://docs.google.com/presentation/d/1uM-zgSiAV3gBWJdqmswxKKJ7hCjIPgc8Ow3252oAVig/edit#slide=id.p4
While the overwhelming way to edit genome, CRISPR, allows human beings to finish permanent modification of genes within organisms, this new ability is also attracting people to explore the ways. People have used four families of engineered nucleases. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
In 2nd May, 2016, an article named DNA-guided genome editing using NgAgo (the Natronobacterium gregoryi Argonaute) came up with a new way to edit genome. Since it was first published in Nature Biotech in May 2016, NgAgo has received much attention and the plasmid has been requested by labs around the globe nearly 400 times.
Argonautes a nuclease, which exists in the bacteria, plants, fungi, mammalian cells.
Recently found TtAgo acquired from bacterial Argonaute capable of binding to single-stranded DNA and the latter as a guide to degrade DNA plasmids. This phenomenon prompted Argonaute certain species can be used as internal DNA endonuclease.
Like the CRISPR/Cas system, NgAgo was reported to be suitable for genome editing, but this has not been replicated.
Today, I gonna talk around this sentence. The third sentence appears on the Wikipedia page of NgAgo.
Like the CRISPR/Cas system
As aimed to the same target with CRISPR, NgAgo has the similarity with CRISPR in many ways, but it also has its own advantages.
Here is a side-by-side comparison of the two systems, based on data presented in the NBT paper as well as the characteristics of each component in the gene editing systems.
1. Comparable gene editing efficiency
2. Higher fidelity (less off-target effect)
Data showed that NgAgo resulted in low guide–target mismatch and high efficiency when editing difficult templates, such as GC-rich genomic targets. Because the cells themselves produce large amounts of RNA fragments, Cas9 have with these non-specific RNAs binding possibility misguided cutting non-targeting of genomic location; and Cas9 to guide RNA and DNA sequences targeting high mismatch tolerance (5 nucleotide mismatches will not stop cutting), so there is a certain off-target Cas9 defects.
Unlike Cas9 only contains 19 bases, the gDNA combined with NgAgo contains 24 bases. These 5 more bases, theoretically, can promote the accuracy 4 of 5th power, that is 1024 times.
3. Fewer sequence constraints (no need for PAM)
NgAgo did not seem to require a protospacer-adjacent motif (PAM), making it easier to use for targeted genome editing, which gives a researcher unprecedented freedom to target any sequence of DNA.
4. Guides are readily available (5’p ssDNA oligo could be purchased at low cost)
Using 5’ phosphorylated ssDNAs as guide molecules reduces the possibility of cellular oligonucleotides misleading NgAgo. A guide molecule can only be attached to NgAgo during the expression of the protein. Once the guide is loaded, NgAgo cannot swap free floating ssDNA for its gDNA.
5. Guides are of higher stability (DNA as compared to RNA in CRISPR/Cas9 system)
NgAgo target specificity is dictated by a DNA guide sequence rather than an RNA guide as with Cas9. Since the formation of RNA secondary structure easier, so rich gene sequence GC, Cas9 system inefficient
While both the CRISPR-Cas9 and NgAgo genome engineering methods have their own pros and cons.
1. Unlike an RNA guide, it cannot be produced from a plasmid in cells.
2. Additionally, in vitro assembly of NgAgo/ssDNA requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes the endonuclease activity of the enzyme.
3. In vivo, only nascent NgAgo protein (probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene in vivo.
NgAgo was reported to be suitable for genome editing，
The technology nuclease specific sites of the genome mutations, insertions, substitutions and other transformation. Gene editing process includes three steps:
First, a specially designed wizard DNA or RNA (gene-oriented element) and endonuclease (Gene cutting element) binding, here is gDNA, which first led to a specific area targeted gene;
Secondly, the nucleic acid endonuclease gene targeting specific areas for cutting, each on two DNA to form a gap, resulting in double-strand break (DSB). Here use argonaute endonuclease enzyme
Finally, the activation of innate intracellular DSB repair mechanisms of this phenomenon, the repair gap At the same time can be mutations, insertions, substitutions and other transformation.
“one-guide faithful” rule
guides can only be loaded when NgAgo protein is in the process of expression
NgAgo + 5’p ssDNA = NgAgo/gDNA
(Protein) (Guide)
NgAgo/gDNA + target DNA → Gene Editing
5 'end phosphorylated single-stranded DNA as a guide wizard, Argonaute nuclease combined with the Wizard DNA, for DNA complementary to the target DNA wizard cutting, resulting in the targeting DNA double-strand breaks.
Enzyme is a nucleic acid capable of Argonaute 5 'phosphorylated end of single-stranded DNA as short-chain double-stranded DNA in a targeting guide at 10-50 ° C under endonuclease cleavage of double-strand breaks caused by Argonaute nucleic acids.
After targeting genomic DNA double strand breaks caused in the presence of exogenous DNA fragments with homologous sequences of case, by the cell's own repair pathways HomologousDirectedRepair the outer source DNA fragment into double-strand breaks area, so as to achieve the targeted gene or functional areas according to the design change.
The engineered NLS-NgAgo was transfected to Hela cells and it shows that the expressed NLS-NgAgo was compartmented in the nucleus (DAPI+) and the cells maintained normal morphology.
The NgAgo replication experiments have been conducted by scientists from around the world.
US, Europe, Australia, China, Japan, India
The popularity of the experiments about NgAgo also arose after the NBT article has been published. Labs and scientists are trying to repeat this new method.
Scientists have really worked hard for NgAgo, many well-established detection methods for genome editing have been utilized.
Detection Methods for Genome Editing
· T7E1
· Surveyor
· Sanger sequencing
· NGS
· Phenotype detection
And other validated assays for genome editing
NgAgo has been tested in many model systems
Systems tested
· Mammalian cell lines (HEK293, Hela, etc)
· Mouse/human NS cells
· Yeast
· Fruit fly
· C. elegans
· Zebra fish
· Mouse zygote
And, maybe even more…..
but this has not been replicated.
This is a global experimentation on NgAgo for genome editing; scientists from many countries have contributed budget, time and effort for it.
A group of scientists has been test NgAgo on Zebrafish, but the result from their experiments just shows that the gDNA/NgAgo system provides an alternative strategy for gene knockdown in zebrafish, but not edit the genome.
Trying to find out which part is wrong, some also analyzed the NBT paper. They got the logic in this paper in this chart.
E. coli expressed and purified GST-NgAgo needs 2 guides to linearize plasmid
Mammalian expressed and purified FLAG-NgAgo-HA only needs 1 guide to linearize plasmid
in vitro, E. coli GST-NgAgo needs 2 guides
in vitro, Mammalian FLAG-NgAgo-HA needs only 1 guide
So in their opinion there is a jump from 2 guids to 1 guide.
Han also added A general protocol of NgAgo/gDNA-mediated genome editing trying to help for the experiments.
In his papers, he mentioned these 3 parts.
Cell culture
Transfection
Genomic DNA extraction
In conclusion,
NgAgo will have to go through a couple years of validation to inspect its safety profile. It will be interesting to see how this new system matures over time.
Sources
https://en.wikipedia.org/wiki/NgAgo
https://www.nature.com/nbt/journal/v34/n7/fig_tab/nbt.3547_SF4.html
https://www.nature.com/cr/journal/v26/n12/full/cr2016134a.html
http://www.nature.com/nbt/journal/v34/n7/full/nbt.3547.html#correction1
https://medium.com/@GenomicsEagles/the-global-experimentation-on-ngago-4f0e282c2190
https://medium.com/@GenomicsEagles/deconstructing-ngago-2c2fbf272658
http://www.nature.com/news/replications-ridicule-and-a-recluse-the-controversy-over-ngago-gene-editing-intensifies-1.20387
http://blog.addgene.org/google-forums-round-up-first-impressions-of-ngago
http://baike.baidu.com/link?url=VOK2jGUOfvIAIbIFFDFQl3I52SfMDeipDhYYqg3ujMbrhHzLT3Y-wpdaybmYyyNqJlingJeFGDY76VMj6g2pRgcEdl4090h5X9MffbtyeItjbkze4t38pFYmGB81DNp2AhMlfgFO0SRTLdvp2623Xa